Previous work in our laboratory and by others, has demonstrated the complexity of regulatory networks that coordinately control the expression of virulence factors in P. aeruginosa. We have used genome-wide transposon mutant analyses to define the functions of two-component regulatory systems and enzymes involved in synthesis or degradation of second messenger c-diGMP. One of the consistent features of phenotypes of strains containing mutations in these regulators is the inverse relationship between expression of genes involved in production and secretion of toxins and those responsible for biofilm formation. By analyzing the phenotypes of a set of mutants with insertions in genes encoding diguanylate cyclases and phosphodiesterases (the DDGEF and EAL domain-containing proteins) we identified a subset of six genes that control the expression of virulence factors. A detailed analysis of these novel virulence regulators will be the subject of the proposed work. This research plan will focus on establishing the role of c-diGMP production and metabolism in the expression of various virulence traits by generating active site mutants in the diguanylate cyclases and phosphodiesterases, which we have recently identified as regulators of virulence. A proteome-wide screen will be undertaken to identify c-diGMP binding proteins that serve as adapters for transmission of signals between diguanylate cyclases and effectors resulting in regulation of virulence genes. Finally, we will undertake a detailed analysis of the novel three components RocS1/RocR/ RocA1 signal transduction system in order to determine the mechanism of action of signaling domains in global transcriptional responses involving the second messenger c-diGMP. This work will be the basis of improving our understanding of the molecular circuitry, which assimilates and accurately transduces environmental stimuli, allowing pathogens to respond to and thrive in the environment of the infected host.